The present invention relates to a class of substituted triazolo-pyridazine derivatives and to their use in therapy. More particularly, this invention is concerned with substituted 1,2,4-triazolo[4,3-b]pyridazine derivatives which are ligands for GABAA receptors and are therefore useful in the therapy of deleterious mental states.
Receptors for the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), are divided into two main classes: (1) GABAA receptors, which are members of the ligand-gated ion channel superfamily; and (2) GABAB receptors, which may be members of the G-protein linked receptor superfamily. Since the first cDNAs encoding individual GABAA receptor subunits were cloned the number of known members of the mammalian family has grown to include at least six xcex1 subunits, four xcex2 subunits, three xcex3 subunits, one xcex4 subunit, one xcex5 subunit and two xcfx81 subunits.
Although knowledge of the diversity of the GABAA receptor gene family represents a huge step forward in our understanding of this ligand-gated ion channel, insight into the extent of subtype diversity is still at an early stage. It has been indicated that an xcex1 subunit, a xcex2 subunit and a xcex3 subunit constitute the minimum requirement for forming a fully functional GABAA receptor expressed by transiently transfecting cDNAs into cells. As indicated above, xcex4, xcex5 and xcfx81 subunits xcex11 so exist, but are present only to a minor extent in GABAA receptor populations.
Studies of receptor size and visualisation by electron microscopy conclude that, like other members of the ligand-gated ion channel family, the native GABAA receptor exists in pentameric form. The selection of at least one xcex1, one xcex2 and one xcex3 subunit from a repertoire of seventeen xcex1allows for the possible existence of more than 10,000 pentameric subunit combinations. Moreover, this calculation overlooks the additional permutations that would be possible if the arrangement of subunits around the ion channel had no constraints (i.e. there could be 120 possible variants for a receptor composed of five different subunits).
Receptor subtype assemblies which do exist include, amongst many others, xcex11xcex22xcex32, xcex12xcex22/3xcex32, xcex13xcex2xcex32/3, xcex12xcex2xcex31, xcex15xcex23xcex32/3, xcex16xcex2xcex32, xcex16xcex2xcex4 and xcex14xcex2xcex4. Subtype assemblies containing an xcex11 subunit are present in most areas of the brain and are thought to account for over 40% of GABAA receptors in the rat. Subtype assemblies containing xcex12 and xcex13 subunits respectively are thought to account for about 25% and 17% of GABAA receptors in the rat. Subtype assemblies containing an xcex15 subunit are expressed predominantly in the hippocampus and cortex and are thought to represent about 4% of GABAA receptors in the rat.
A characteristic property of all known GABAA receptors is the presence of a number of modulatory sites, one of which is the benzodiazepine (BZ) binding site. The BZ binding site is the most explored of the GABAA receptor modulatory sites, and is the site through which anxiolytic drugs such as diazepam and temazepam exert their effect. Before the cloning of the GABAA receptor gene family, the benzodiazepine binding site was historically subdivided into two subtypes, BZ1 and BZ2, on the basis of radioligand binding studies. The BZ1 subtype has been shown to be pharmacologically equivalent to a GABAA receptor comprising the xcex11 subunit in combination with a xcex2 subunit and xcex32. This is the most abundant GABAA receptor subtype, and is believed to represent almost half of all GABAA receptors in the brain.
Two other major populations are the xcex12xcex2xcex32 and xcex13xcex2xcex32/3 subtypes. Together these constitute approximately a further 35% of the total GABAA receptor repertoire. Pharmacologically this combination appears to be equivalent to the BZ2 subtype as defined previously by radioligand binding, although the BZ2 subtype may also include certain xcex15-containing subtype assemblies. The physiological role of these subtypes has hitherto been unclear because no sufficiently selective agonists or antagonists were known.
It is now believed that agents acting as BZ agonists at xcex11xcex2xcex32, xcex12xcex2xcex32 or xcex13xcex2xcex32 subunits will possess desirable anxiolytic properties. Compounds which are modulators of the benzodiazepine binding site of the GABAA receptor by acting as BZ agonists are referred to hereinafter as xe2x80x9cGABAA receptor agonistsxe2x80x9d. The xcex11-selective GABAA receptor agonists alpidem and zolpidem are clinically prescribed as hypnotic agents, suggesting that at least some of the sedation associated with known anxiolytic drugs which act at the BZ1 binding site is mediated through GABAA receptors: containing the xcex11 subunit. Accordingly, it is considered that GABAA receptor agonists which interact more favourably with the xcex12 and/or xcex13 subunit than with xcex11 will be effective in the treatment of anxiety with a reduced propensity to cause sedation. Also, agents which are antagonists or inverse agonists at xcex11 might be employed to reverse sedation or hypnosis caused by xcex11 agonists.
The compounds of the present invention, being selective ligands for GABAA receptors, are therefore of use in the treatment and/or prevention of a variety of disorders of the central nervous system. Such disorders include anxiety disorders, such as panic disorder with or without agoraphobia, agoraphobia without history of panic disorder, animal and other phobias including social phobias, obsessive-compulsive disorder, stress disorders including post-traumatic and acute stress disorder, and generalized or substance-induced anxiety disorder; neuroses; convulsions; migraine; depressive or bipolar disorders, for example single-episode or recurrent major depressive disorder, dysthymic disorder, bipolar I and bipolar II manic disorders, and cyclothymic disorder; psychotic disorders including schizophrenia; neurodegeneration arising from cerebral ischemia; attention deficit hyperactivity disorder; and disorders of circadian rhythm, e.g. in subjects suffering from the effects of jet lag or shift work.
Further disorders for which selective ligands for GABAA receptors may be of benefit include pain and nociception; emesis, including acute, delayed and anticipatory emesis, in particular emesis induced by chemotherapy or radiation, as well as post-operative nausea and vomiting; eating disorders including anorexia nervosa and bulimia nervosa; premenstrual syndrome; muscle spasm or spasticity, e.g. in paraplegic patients; and hearing loss. Selective ligands for GABAA receptors may also be effective as pre-medication prior to anaesthesia or minor procedures such as endoscopy, including gastric endoscopy.
In DE-A-2741763, and in U.S. Pat. Nos. 4,260,755, 4,260,756 and 4,654,343, are described various classes of 1,2,4-triazolo[4,3-b]pyridazine derivatives which are alleged to be useful as anxiolytic agents. The compounds described in DE-A-2741763 and in U.S. Pat. Nos. 4,260,755 and 4,654,343 possess a phenyl substituent at the 6-position of the triazolo-pyridazine ring system. The compounds described in U.S. Pat. No. 4,260,756, meanwhile, possess a heteroaryl moiety at the 6- or 8-position. In none of these publications, however, is there any disclosure or suggestion of 1,2,4-triazolo[4,3-b]pyridazine derivatives wherein the substituent at the 6-position is attached through a directly linked oxygen atom.
EP-A-0085840 and EP-A-0134946 describe related series of 1,2,4-triazolo[3,4-a]phthalazine derivatives which are stated to possess antianxiety activity. However, there is no disclosure nor any suggestion in either of these publications of replacing the benzo moiety of the triazolo-phthalazine ring system with any other functionality.
The present invention provides a class of triazolo-pyridazine derivatives which possess desirable binding properties at various GABAA receptor subtypes. The compounds in accordance with the present invention have good affinity as ligands for the xcex12 and/or xcex13 subunit of the human GABAA receptor. The compounds of this invention may interact more favourably with the xcex12 and/or xcex13 subunit than with the xcex11 subunit. Desirably, the compounds of the invention will exhibit functional selectivity in terms of a selective efficacy for the xcex12 and/or xcex13 subunit relative to the xcex11 subunit.
The compounds of the present invention are GABAA receptor subtype ligands having a binding affinity (Ki) for the xcex12 and/or xcex13 subunit, as measured in the assay described hereinbelow, of 100 nM or less, typically of 50 nM or less, and ideally of 10 nM or less. The compounds in accordance with this invention may possess at least a 2-fold, suitably at least a 5-fold, and advantageously at least a 10-fold, selective affinity for the xcex12 and/or xcex13 subunit relative to the xcex11 subunit. However, compounds which are not selective in terms of their binding affinity for the xcex12 and/or xcex13 subunit relative to the xcex11-subunit are also encompassed within the scope of the present invention; such compounds will desirably exhibit functional selectivity in terms of a selective efficacy for the xcex12 and/or xcex13 subunit relative to the xcex11 subunit. Moreover, the compounds according to the present invention possess interesting pharmacokinetic properties, notably in terms of improved oral bioavailability.
The present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof: 
wherein
Z represents trifluoromethyl, 2-methylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-fluorobut-3-enyl, cyclobutyl, 1-methylcyclobutyl, 1-fluorocyclobutyl, 3-fluorocyclobutyl, 3,3-difluorocyclobutyl, 3-hydroxycyclobutyl, 3-benzyloxycyclobutyl, 3-oxocyclobutyl, 1-methylcyclohexyl, 4,4-difluoro-1-methylcyclohexyl, cyclopentylmethyl, 4-fluorocyclohex-3-enyl, 3-fluorophenyl, tetrahydrofur-2-yl, pyrrolidin-1-yl, 4-methyltetrahydropyran-4-yl or thien-2-yl;
R1 represents hydrogen or fluoro; and
R2 represents methyl-isoxazolyl, methyl-pyrazolyl, methyl-imidazolyl, benzimidazolyl or methyl-triazolyl;
provided that, when Z represents 1-methylcyclobutyl, R1 is hydrogen and R2 represents 1-methyl-1H-1,2,4-triazol-3-yl or 2-methyl-2H-1,2,4-triazol-3-yl, then the fluorine atom is not at the 2-position of the phenyl ring.
Certain compounds in accordance with the present invention are encompassed within the generic scope of co-pending International Patent Application No. PCT/GB97/01946, published on Feb. 5, 1998 as WO 98/04559. There is, however, no specific disclosure therein of compounds corresponding to those of formula I as defined above.
The present invention also provides a compound of formula I as depicted above, or a pharmaceutically acceptable salt thereof, wherein
Z represents trifluoromethyl, 2-methylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-fluorobut-3-enyl, cyclobutyl, 1-methylcyclobutyl, 1-fluorocyclobutyl, 3-fluorocyclobutyl, 3-hydroxycyclobutyl, 3-benzyloxycyclobutyl, 1-methylcyclohexyl, cyclopentylmethyl, pyrrolidin-1-yl or thien-2-yl; and
R1 and R2 are as defined above; provided that, when Z represents 1-methylcyclobutyl, R1 is hydrogen and R2 represents 1-methyl-1H-1,2,4-triazol-3-yl or 2-methyl-2H-1,2,4-triazol-3-yl, then the fluorine atom is not at the 2-position of the phenyl ring.
The present invention further provides a compound of formula I as depicted above, or a pharmaceutically acceptable salt thereof, wherein
Z represents cyclobutyl, 1-methylcyclobutyl, 1-fluorocyclobutyl, 1-methylcyclohexyl, pyrrolidin-1-yl or thien-2-yl; and
R1 and R2 are as defined above;
provided that, when Z represents 1-methylcyclobutyl, R1 is hydrogen and R2 represents 1-methyl-1H-1,2,4-triazol-3-yl or 2-methyl-2H-1,2,4-triazol-3-yl, then the fluorine atom is not at the 2-position of the phenyl ring.
For use in medicine, the salts of the compounds of formula I will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
In the compounds of formula I above, the moiety Z suitably represents cyclobutyl.
The substituent R2 suitably represents a ring of structure (a), (b), (c), (d), (e), (f) or (g): 
where the asterisk * denotes the point of attachment of the ring to the remainder of the molecule.
A particular moiety R2 is represented by the structure (g) as depicted above.
Where permissible, the compounds of formula I as defined above suitably possess a fluorine atom at the 2-position of the phenyl ring.
A particular sub-class of compounds according to the invention is represented by the compounds of formula IIA, and pharmaceutically acceptable salts thereof. 
wherein R1 is as defined above; and
R3 represents hydrogen or fluoro.
Suitably, R3 represents hydrogen.
A particular subset of the compounds of formula IIA above is represented by the compounds of formula IIB, and pharmaceutically acceptable salts thereof: 
wherein R1 and R3 are as defined above.
Specific compounds within the scope of the present invention include:
7-cyclobutyl-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2-fluorophenyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(3-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(4-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2,4-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(3,5-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2,4-difluorophenyl)-7-(1-methylcyclobutyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(3,4-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2,3-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b ]pyridazine;
7-cyclobutyl-3-(2,6-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2,5-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2,4-difluorophenyl)-7-(1-methylcyclohexyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2,4-difluorophenyl)-7-(1-methylcyclohexyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2-fluorophenyl)-6-(1-methyl-1H-pyrazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2-fluorophenyl)-6-(5-methylisoxazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2-fluorophenyl)-6-(1-methyl-1H-imidazol 2-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclobutyl-3-(2-fluorophenyl)-6-(4-methyl-4H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-7-(thien-2-yl)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2,4-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-7-(thien-2-yl)-1,2,4-triazolo[4,3-b]pyridazine;
6-(1H-benzimidazol-2-ylmethoxy)-7-cyclobutyl-3-(2,4-difluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2,4-difluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2,4-difluorophenyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-7-(pyrrolidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-7-(pyrrolidin-1-yl)-1,2,4-triazolo [4,3-b]pyridazine;
7-cyclobutyl-3-(2-fluorophenyl)-6-(1-methyl-1H-imidazol-4-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-(1-fluorocyclobutyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo [4,3-b]pyridazine;
7-cyclobutyl-3-(2-fluorophenyl)-6-(2-methyl-2H-pyrazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-(2,2-dimethylpropyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo [4,3-b]pyridazine;
3-(2-fluorophenyl)-7-(2-methylpropyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-7-(3-methylbutyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-cyclopentylmethyl-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-(3-benzyloxycyclobutyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-
3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-7-(3-hydroxycyclobutyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-(1-fluorobut-3-enyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-(3-fluorocyclobutyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-7-trifluoromethyl-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-7-(4-methyltetrahydropyran-4-yl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-7-(4-methyltetrahydropyran-4-yl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo [4,3-b]pyridazine;
7-(4,4-difluoro-1-methylcyclohexyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-(4-fluoro-1-methylcyclohex-3-enyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
7-(4,4-difluoro-1-methylcyclohexyl)-3-(2-fluorophenyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-7-(3-oxocyclobutyl)-1,2,4-triazolo [4,3-b]pyridazine;
7-(3,3-difluorocyclobutyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-7-(tetrahydrofur-2-yl)-1,2,4-triazolo[4,3-b]pyridazine;
7-(3-fluorophenyl)-3-(2-fluorophenyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;
and pharmaceutically acceptable salts thereof.
Also provided by the present invention is a method for the treatment and/or prevention of anxiety which comprises administering to a patient in need of such treatment an effective amount of a compound of formula I as defined above or a pharmaceutically acceptable salt thereof.
Further provided by the present invention is a method for the treatment and/or prevention of convulsions (e.g. in a patient suffering from epilepsy or a related disorder) which comprises administering to a patient in need of such treatment an effective amount of a compound of formula I as defined above or a pharmaceutically acceptable salt thereof.
The binding affinity (Ki) of the compounds according to the present invention for the xcex13 subunit of the human GABAA receptor is conveniently as measured in the assay described hereinbelow. The xcex13 subunit binding affinity (Ki) of the compounds of the invention is ideally 10 nM or less, preferably 2 nM or less, and more preferably 1 nM or less.
The compounds according to the present invention will ideally elicit at least a 40%, preferably at least a 50%, and more preferably at least a 60%, potentiation of the GABA EC20 response in stably transfected recombinant cell lines expressing the xcex13 subunit of the human GABAA receptor. Moreover, the compounds of the invention will ideally elicit at most a 30%, preferably at most a 20%, and more preferably at most a 10%, potentiation of the GABA EC20 response in stably transfected recombinant cell lines expressing the xcex11 subunit of the human GABAA receptor.
The potentiation of the GABA EC20 response in stably transfected cell lines expressing the xcex13 and xcex11 subunits of the human GABAA receptor can conveniently be measured by procedures analogous to the protocol described in Wafford et al., Mol. Pharmacol., 1996, 50, 670-678. The procedure will suitably be carried out utilising cultures of stably transfected eukaryotic cells, typically of stably transfected mouse Ltk fibroblast cells.
The compounds according to the present invention exhibit anxiolytic activity, as may be demonstrated by a positive response in the elevated plus maze and conditioned suppression of drinking tests (cf. Dawson et al., Psychopharmacology, 1995, 121, 109-117). Moreover, the compounds of the invention are substantially non-sedating, as may be confirmed by an appropriate result obtained from the response sensitivity (chain-pulling) test (cf. Bayley et al., J. Psychopharmacol., 1996, 10, 206-213).
The compounds according to the present invention may also exhibit anticonvulsant activity. This can be demonstrated by the ability to block pentylenetetrazole-induced seizures in rats and mice, following a protocol analogous to that described by Bristow et al. in J. Pharmacol. Exp. Ther., 1996, 279, 492-501.
In order to elicit their behavioural effects, the compounds of the invention will ideally be brain-penetrant; in other words, these compounds will be capable of crossing the so-called xe2x80x9cblood-brain barrierxe2x80x9d. Preferably, the compounds of the invention will be capable of exerting their beneficial therapeutic action following administration by the oral route.
The invention also provides pharmaceutical compositions comprising one or more compounds of this invention in association with a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Typical unit dosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
In the treatment of anxiety, a suitable dosage level is about 0.01 to 250 mg/kg per day, preferably about 0.05 to 100 mg/kg per day, and especially about 0.05 to 5 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day.
The compounds of formula I as defined above may be prepared by a process which comprises reacting a compound of formula III with a compound of formula IV: 
wherein Z, R1 and R2 are as defined above; and L1 represents a suitable leaving group.
The leaving group L1 is typically a halogen atom, especially chloro.
The reaction between compounds III and IV is conveniently effected by stirring the reactants in a suitable solvent, typically N,N-dimethyl-formamide or tetrahydrofuran, in the presence of a strong base such as sodium hydride, lithium bis(trimethylsilyl)amide or potassium bis(trimethylsilyl)amide.
The intermediates of formula III above may be prepared by reacting a compound of formula V with a substantially equimolar amount of a hydrazine derivative of formula VI: 
wherein Z, R1 and L1 are as defined above, and L2 represents a suitable leaving group; followed, if necessary, by separation of the resulting mixture of isomers by conventional means.
The leaving group L2 is typically a halogen atom, especially chloro. In the intermediates of formula V, the leaving groups L1 and L2 may be the same or different, but are suitably the same, preferably both chloro.
The reaction between compounds V and VI is conveniently effected by heating the reactants in the presence of a proton source such as triethylamine hydrochloride, typically at reflux in an inert solvent such as xylene or 1,4-dioxane.
Alternatively, the intermediates of formula III above may be prepared by reacting a hydrazine derivative of formula VII with an aldehyde derivative of formula VIII: 
wherein Z, R1 and L1 are as defined above; followed by cyclization of the intermediate Schiff""s base thereby obtained.
The reaction between compounds VII and VIII is conveniently effected under acidic conditions, for example in the presence of a mineral acid such as hydrochloric acid. Cyclization of the resulting Schiff""s base intermediate may then conveniently be carried out by treatment with iron(III) chloride in a suitable solvent, e.g. an alcoholic solvent such as ethanol, at an elevated temperature, typically at a temperature in the region of 60-70xc2x0 C.
The intermediates of formula VII above may be prepared by reacting the appropriate compound of formula V as defined above with hydrazine hydrate, typically in 1,4-dioxane at the reflux temperature of the solvent; followed, if necessary, by separation of the resulting mixture of isomers by conventional means.
In an alternative approach, the intermediates of formula III above may be prepared by reacting the hydrazine derivative of formula VII as defined above with a compound of formula IX: 
wherein R1 is as defined above, and Q represents a reactive carboxylate moiety; followed by cyclization of the hydrazide derivative of formula X thereby obtained: 
wherein Z, R1 and L1 are as defined above.
Suitable values for the reactive carboxylate moiety Q include esters, for example C1-4 alkyl esters; acid anhydrides, for example mixed anhydrides with C1-4 alkanoic acids; acid halides, for example acid chlorides; and acylimidazoles. Suitably, Q represents an acid chloride moiety.
The reaction between compounds VII and IX is conveniently effected under basic conditions, e.g. in the presence of triethylamine, suitably in an inert solvent such as diethyl ether, and typically at a temperature in the region of 0xc2x0 C. Cyclization of the resulting compound of formula X may then conveniently be carried out by treatment with 1,2-dibromo-1,1,2,2-tetrachloroethane and triphenylphosphine, in the presence of a base such as triethylamine, suitably in an inert solvent such as acetonitrile, and typically at a temperature in the region of 0xc2x0 C.
The reaction between compound V and hydrazine hydrate or compound VI will, as indicated above, usually give rise to a mixture of isomeric products depending upon whether the hydrazine nitrogen atom displaces the leaving group L1 or L2. Thus, in addition to the required product of formula III, the isomeric compound wherein the moiety Z is attached at the 8-position will usually be obtained to some extent; and likewise for compound VII. For this reason it will generally be necessary to separate the resulting mixture of isomers by conventional methods such as chromatography.
In another procedure, the compounds of formula I as defined above may be prepared by a process which comprises reacting a compound of formula XI (or its 1,2,4-triazolo[4,3-b]pyridazin-6-one tautomer) with a compound of formula XII: 
wherein Z, R1 and R2 are as defined above, and L3 represents a suitable leaving group.
The leaving group L3 is suitably a halogen atom, typically chloro or bromo.
The reaction between compounds XI and XII is conveniently effected by stirring the reactants in a suitable solvent, typically N,N-dimethylformamide, in the presence of a strong base such as sodium hydride.
The intermediates of formula XI above may conveniently be prepared by reacting a compound of formula III as defined above with an alkali metal hydroxide, e.g. sodium hydroxide. The reaction is conveniently effected in an inert solvent such as aqueous 1,4-dioxane, ideally at the reflux temperature of the solvent.
In a further procedure, the compounds of formula I as defined above may be prepared by a process which comprises reacting a compound of formula Zxe2x80x94CO2H with a compound of formula XIII: 
wherein Z, R1 and R2 are as defined above; in the presence of silver nitrate and ammonium persulphate.
The reaction is conveniently carried out in a suitable solvent, for example water or aqueous acetonitrile, optionally under acidic conditions, e.g. using sulphuric acid, typically at an elevated temperature.
The intermediates of formula XIII correspond to the compounds of formula I as defined above wherein Z is hydrogen, and they may therefore be prepared by methods analogous to those described above for preparing the corresponding compounds of formula I.
In a still further procedure, the compounds of formula I as defined above may be prepared by a process which comprises reacting a compound of formula XIV with a compound of formula XV: 
wherein Z, R1 and R2 are as defined above, M represents xe2x80x94B(OH)2 or xe2x80x94Sn(Alk)3 in which Alk represents a C1-6 alkyl group, typically n-butyl, and L4 represents a suitable leaving group; in the presence of a transition metal catalyst.
The leaving group L4 is suitably a halogen atom, e.g. bromo.
A suitable transition metal catalyst of use in the reaction between compounds XIV and XV comprises dichlorobis(triphenylphosphine)-palladium(II) or tetrakis(triphenylphosphine)palladium(0).
The reaction between compounds XIV and XV is conveniently effected in an inert solvent such as N,N-dimethylformamide, typically at an elevated temperature.
The intermediates of formula XIV may be prepared by reacting a compound of formula IV as defined above with a compound of-formula XVI: 
wherein Z, L1 and L4 are as defined above; under conditions analogous to those described above for the reaction between compounds III and IV.
In a yet further procedure, the compounds of formula I wherein Z represents 1-fluorocyclobutyl may be prepared by a process which comprises reacting a compound of formula XVII: 
wherein R1 and R2 are as defined above; with a fluorinating agent.
Similarly, the compounds of formula I wherein Z represents 3-fluorocyclobutyl, or 1-fluorobut-3-enyl, or a mixture thereof, may be prepared by a process which comprises reacting a compound of formula XVIII: 
wherein R1 and R2 are as defined above; with a fluorinating agent. Where a mixture of products is obtained, the individual components thereof may be isolated by conventional means including chromatography.
Similarly, the compounds of formula I wherein Z represents 3,3-difluorocyclobutyl may be prepared by a process which comprises reacting the corresponding compound wherein Z represents 3-oxocyclobutyl with a fluorinating agent.
A suitable fluorinating agent for use in the above reactions is diethylaminosulphur trifluoride (DAST), in which case the reaction can conveniently be brought about by stirring the reactants in an inert solvent such as dichloromethane, typically at a temperature in the region of xe2x88x9278xc2x0 C.
The intermediates of formula XVII may be prepared by reacting a compound of formula IV as defined above with a compound of formula XIX: 
wherein R1 and L1 are as defined above; under conditions analogous to those described above for the reaction between compounds III and IV.
The intermediates of formula XIX may in turn be prepared by reacting cyclobutanone with a compound of formula XX: 
wherein R1 and L1 are as defined above, and Alk represents C1-6 alkyl, typically methyl.
The reaction is conveniently effected by treating the reagents with a fluoride source, e.g. a catalytic quantity of tetrabutylammonium difluorotriphenylstannate, suitably in an inert solvent such as tetrahydrofuran.
The intermediates of formula XX correspond to the compounds of formula III as defined above wherein Z is xe2x80x94Si(Alk)3, and they may therefore be prepared by methods analogous to those described above for preparing the corresponding compounds of formula III.
The compounds of formula XVIII above (corresponding to compounds of formula I wherein Z represents 3-hydroxycyclobutyl) may be prepared by hydrogenolysis of a compound of formula XXI: 
wherein R1 and R2 are as defined above.
The reaction is conveniently effected by transfer hydrogenation, which comprises contacting compound XXI with a hydrogenation catalyst in the presence of a hydrogen donor. A suitable hydrogenation catalyst is palladium on carbon, ideally 10% palladium on carbon. A suitable hydrogen donor is ammonium formate, in which case the reaction is advantageously performed in formic acid.
In an additional procedure, the compounds of formula I as defined above may be prepared by a process which comprises reacting a compound of formula XXII with a compound of formula XXIII: 
wherein Z, R1 and R2 are as defined above, L5 represents a suitable leaving group, and E represents xe2x80x94B(OH)2 or the residue of an organozinc reagent; in the presence of a transition metal catalyst.
The leaving group L5 is suitably a halogen atom, e.g. bromo or iodo.
Where E represents xe2x80x94B(OH)2, the transition metal catalyst of use in the reaction between compounds XXII and XXIII is suitably tetrakis(triphenylphosphine)palladium(0), and the reaction is conveniently effected at an elevated temperature in the presence of potassium phosphate and a solvent such as N,N-dimethylformamide.
Where E represents the residue of an organozinc reagent, the intermediate XXIII is suitably prepared by reacting an iodide derivative Zxe2x80x94I with zinc dust, typically in the presence of 1,2-dibromoethane and a solvent such as N,N-dimethylformamide. In this instance, the transition metal catalyst of use in the reaction between compounds XXII and XXIII is ideally tris(dibenzylideneacetone)dipalladium(0), and the reaction is conveniently effected in the presence of tri-2-furylphosphine and a solvent such as N,N-dimethylformamide.
The compounds of formula XXI above (corresponding to compounds of formula I wherein Z represents 3-benzyloxycyclobutyl) may be similarly prepared by reacting a compound of formula XXII as defined above with a compound of formula XXIV: 
wherein E is as defined above; in the presence of a transition metal catalyst; under conditions analogous to those described above for the reaction between compounds XXII and XXIII.
The intermediates of formula XXII may be prepared by reacting a compound of formula IV as defined above with a compound of formula XXV: 
wherein R1, L1 and L5 are as defined above; under conditions analogous to those described above for the reaction between compounds III and IV.
The intermediates of formula XXV may suitably be prepared by treatment of the appropriate precursor of formula XX as defined above with a fluoride source, e.g. tetrabutylammonium difluorotriphenylstannate or tris(dimethylamino)sulphur (trimethyl)difluoride, in the presence of an L5-containing reagent, e.g. 1,2-dibromotetrafluoroethane or 1,2-diiodoethane.
The compounds of formula I as defined above wherein Z represents trifluoromethyl may be prepared by a process which comprises reacting a compound of formula XXII as defined above with iodotrifluoromethane.
The reaction is suitably performed in the presence of copper powder, typically in a sealed tube at an elevated temperature, e.g. a temperature in the region of 80xc2x0 C.
The intermediates of formula IV above may be prepared by the procedures described in EP-A-0421210, or by methods analogous thereto.
Where they are not commercially available, the starting materials of formula V, VI, VIII, IX, XII, XV, XVI and XXIV may be prepared by methods analogous to those described in the accompanying Examples, or by standard methods well known from the art.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
The following Examples illustrate the preparation of compounds according to the invention.
The compounds in accordance with this invention potently inhibit the binding of [3H]-flumazenil to the benzodiazepine binding site of human GABAA receptors containing the xcex12 or xcex13 subunit stably expressed in Ltk cells.
Reagents
Phosphate buffered saline (PBS).
Assay buffer: 10 mM KH2PO4, 100 mM KCl, pH 7.4 at room temperature.
[3H]-Flumazenil (18 nM for xcex11xcex23xcex32 cells; 18 nM for xcex12xcex23xcex32 cells; 10 nM for xcex13xcex23xcex32 cells) in assay buffer.
Flunitrazepam 100 xcexcM in assay buffer.
Cells resuspended in assay buffer (1 tray to 10 ml).
Harvesting Cells
Supernatant is removed from cells. PBS (approximately 20 ml) is added. The cells are scraped and placed in a 50 ml centrifuge tube. The procedure is repeated with a further 10 ml of PBS to ensure that most of the cells are removed. The cells are pelleted by centrifuging for 20 min at 3000 rpm in a benchtop centrifuge, and then frozen if desired. The pellets are resuspended in 10 ml of buffer per tray (25 cmxc3x9725 cm) of cells.
Assay
Can be carried out in deep 96-well plates or in tubes. Each tube contains:
300 xcexcl of assay buffer.
50 xcexcl of [3H]-flumazenil (final concentration for (xcex11xcex23xcex32: 1.8 nM; for xcex12xcex23xcex32: 1.8 nM; for xcex13xcex23xcex32: 1.0 nM).
50 xcexcl of buffer or solvent carrier (e.g. 10% DMSO) if compounds are dissolved in 10% DMSO (total); test compound or flunitrazepam (to determine non-specific binding), 10 xcexcM final concentration.
100 xcexcl of cells.
Assays are incubated for 1 hour at 40xc2x0 C., then filtered using either a Tomtec or Brandel cell harvester onto GF/B filters followed by 3xc3x973 ml washes with ice cold assay buffer. Filters are dried and counted by liquid scintillation counting. Expected values for total binding are 3000-4000 dpm for total counts and less than 200 dpm for non-specific binding if using liquid scintillation counting, or 1500-2000 dpm for total counts and less than 200 dpm for non-specific binding if counting with meltilex solid scintillant. Binding parameters are determined by non-linear least squares regression analysis, from which the inhibition constant Ki can be calculated for each test compound.
The compounds of the accompanying Examples were tested in the above assay, and all were found to possess a Ki value for displacement of [3H]-flumazenil from the xcex12 and/or xcex13 subunit of the human GABAA receptor of 100 nM or less.